Unknown,Transcriptomics,Genomics,Proteomics

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MiRNA-seq of exosomes derived from bone-metastatic lung cancer cells

ABSTRACT: This study aimed to identify miRNAs enriched in exosomes that promote osteolytic bone metastasis. We performed miRNA-seq directly on exosomes purified from the culture supernatants of either non-metastatic (NC-LLC) or bone-metastatic (BM-LLC) Lewis lung carcinoma cells. The goal was to profile the miRNA cargo of these tumor-derived exosomes and identify key regulators of intercellular communication. Integrated analysis with mRNA sequencing data from recipient macrophages identified exosomal miR-484 as a critical driver of osteoclast differentiation and bone metastasis.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mus musculus

SUBMITTER: Yong Zhou

PROVIDER: E-MTAB-15763 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The study focused on the RNA content of supportive (AFT024) and non supportive (BFC012) stromal lines and their respective exosomes to analyze the molecular complexity of the Hematopoietic Stem and Progenitor Cell niche Methods: All the samples from small RNA (SR) and polyA-RNA libraries were sequenced using 1Ã50 bp single reads high-throughput sequencing (RNA-Seq) in single lane on Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks and by Fisher's test for polyA-RNA, or with bowtie and parse tools for smallRNA (galaxy server https://lbcd41.snv.jussieu.fr/). Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm10). We focused on differentially expressed transcripts between i) exosomes from supportive and non supportive stromal cells and ii) exosomes and their respective cells.Using a subtractive strategy, we identified 324 mRNAs and 23 miRNAs specific to the exosomes of the supportive stromal line AFT024. We used gene ontology annotation to study the biological functions associated to these specific gene sets. Transcripts involved in negative regulation of apoptosis were found enriched in AFT exosomes. Conclusions: Our RNA seq analysis combined with biological assays reveal that exosomes serve as an important and novel mediator for the HSPC supporting capacity of stromal cells. This unprecedented effort to resolve the molecular complexity of the HSPC-targeted exosomes, provide new candidat genes of the HSPC support and may help designing innovative stromal-free culture conditions to deliver specific molecules to HSPCs. Fetal liver derived stromal lines and their corresponding secreted exosomes were sequenced using Illumina HiSeq2500 technology (Fasteris, CH). Due to low amount of exosomal RNAs, several RNA extraction were pooled and split to perform 2 technical sequencing replicates that were merged during bionformatic analyses.

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MiRNA-seq of exosomes derived from bone-metastatic lung cancer cells

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